ABSTRACTMacrophages play an essential role in the early immune response against Toxoplasma and are the cell type preferentially infected by the parasite in vivo. Interferon gamma (IFNγ) elicits a variety of anti-Toxoplasma activities in macrophages. Using a genome-wide CRISPR screen we identified ∼130 Toxoplasma genes that determine parasite fitness in naїve macrophages and ∼466 genes that determine fitness in IFNγ-stimulated murine macrophages, seven of which we investigated and confirmed. We show that one of these genes encodes dense granule protein GRA45, which contains a putative chaperone-like domain, and which we show is critical in preventing other GRA effectors from aggregating. Parasites lacking GRA45 mislocalize GRA effectors upon secretion, are more susceptible to IFNγ-mediated growth inhibition, and have reduced virulence in mice. Our results provide a resource for the community to further explore the function of Toxoplasma genes that determine fitness in IFNγ-stimulated macrophages.IMPORTANCEThe intracellular parasite Toxoplasma gondii can cause congenital infections and severe disease in immunocompromised patients. The cytokine IFNγ can block parasite replication by upregulating a variety of toxoplasmacidal mechanisms in many cells, including macrophages. Toxoplasma preferentially infects macrophages. Therefore, the parasite has evolved mechanisms to survive in these cells in the presence of IFNγ. Here, we generated pools of Toxoplasma mutants for every gene and determined which mutants were specifically depleted in IFNγ-stimulated macrophages, thus identifying parasite genes determining fitness in these cells. We show that one of these genes encodes for a dense granule protein (GRA45) that plays an important role in preventing other GRA effectors from aggregating. Parasites without GRA45 mislocalize GRA effectors upon secretion, have enhanced susceptibility to IFNγ-mediated growth inhibition, and are avirulent in mice. Thus, our screen provides a resource to the Toxoplasma community to determine the function of Toxoplasma genes that affect its fitness in IFNγ-stimulated macrophages.